Methods of treating chronic wounds with amniotic fluid having elevated levels of tissue inhibitors of matrix metalloproteinases

ABSTRACT

Methods for treating chronic wounds with an amniotic fluid having a therapeutically effective amount of tissue inhibitors of matrix metalloproteinases (TIMPs) are described. The amniotic fluid can be obtained from a female donor during a period of gestation when the concentration of endogenous TIMPs are at or near their maximum level. The methods described herein are particularly useful for promoting wound healing in chronic wounds having elevated protease activity, such as increased amounts or specific activity of matrix metalloproteinases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is entitled to priority pursuant to 35 U.S.C. §119(e)to U.S. Provisional Patent Application No. 62/127,433, filed Mar. 3,2015, which is herein incorporated by reference in its entirety.

BRIEF SUMMARY OF THE INVENTION

Wound healing is the body's natural response for repairing andregenerating dermal and epidermal tissue. The wound healing process iscomplex, and is generally categorized into four stages: (1)clotting/hemostasis stage; (2) inflammatory stage; (3) tissue cellproliferation stage; and (4) tissue cell remodeling stage.

In general, a chronic wound is a wound that does not heal in apredictable amount of time and/or in the orderly set of stages fortypical wound healing. Chronic wounds may become caught in one or moreof the four states of wound healing, such as remaining in theinflammatory stage for too long, thereby preventing the wound healingprocess from naturally progressing. A chronic wound may also fail toadequately complete one stage of healing before progressing to the next,causing a disruption in the healing process. As another example,epithelialization that occurs during the tissue proliferation stagetypically results in the formation of epithelial cells at the edges ofthe wound, which proliferate over the wound bed to cover it, andcontinue until the cells formed at the various sides of the woundconverge. However, in the case of chronic wounds, this epithelializationprocess may be ineffective, because the epithelial cells may not rapidlyproliferate over the wound bed or respond to this particular stage ofthe wound healing process at all.

Wounds that do not heal completely within approximately three months arealso considered chronic wounds, and in some cases, chronic wounds maynever heal. Such non-healing chronic wounds are often resistant tocommon wound treatment regimens, and many peripheral wounds that do notheal lead to amputation.

Recently, it has been demonstrated that a subset of non-healing chronicwounds are characterized by elevated protease activity (EPA). Inparticular, members of the family of matrix metalloproteinases (MMPs)and other proteases, such as serine proteases (e.g., plasmin, neutrophilelastase, mast cell chymase, etc.) have been shown to inhibit woundhealing when they have high enzymatic activity levels in the wound.Rationalizing that such wounds might respond to a different treatmentregimen than other wounds, point-of-care tests to identify woundscharacterized by EPA have been developed. However, to the best of theknowledge of the inventors, a treatment to inhibit, or otherwise removesuch proteases in EPA wounds for routine use in the clinic has not yetbeen developed.

Methods and treatments that are typically used to promote healing ofchronic wounds and ameliorate symptoms associated with chronic woundsinclude antibiotics and antibacterials; non-steroidal anti-inflammatorydrugs (NSAIDs) and acetaminophen; cleansing, e.g., with sterile water orsterile saline; surgical or mechanical debridement to remove deadtissue, dirt, or other objects that can delay healing and lead toinfection; hyperbaric oxygen therapy (HBO) to increase the amount ofoxygen flow in the body; and topical chemical treatment. Additionally,collagen wound dressings have been used to treat chronic wounds, becausecollagen can be a competing substrate for the proteases that areelevated in chronic wounds, and are thus intended to mitigate theeffects of the elevated protease activity. However, these methods havesome disadvantages in terms of their efficacy.

Accordingly, there exists a need in the art for improved methods andcompositions for treating chronic wounds, and particularly chronicwounds having elevated protease activity.

BRIEF SUMMARY OF THE INVENTION

The invention satisfies this need by providing a method of treatingchronic wounds using human amniotic fluid comprising a therapeuticallyeffective amount of tissue inhibitors of matrix metalloproteinases,which can inhibit protease activity in the wound and thus facilitate thehealing process of otherwise recalcitrant, non-healing chronic wounds.

In one general aspect, the invention relates to a method of treating achronic wound in a subject comprising applying a human amniotic fluid ora processed human amniotic fluid comprising a therapeutically effectiveamount of tissue inhibitors of matrix metalloproteinases to the chronicwound, thereby treating the chronic wound.

According to embodiments of the invention, the human amniotic fluid canbe obtained from a donor during a period of gestation when naturallevels of TIMPs in the amniotic fluid are relatively high, such asbetween week 28 and week 37 of gestation.

In a preferred embodiment, the chronic wound is characterized as havingelevated protease activity. In other preferred embodiments, the chronicwound is an ulcer, e.g., a diabetic foot ulcer, venous leg ulcer, orpressure ulcer.

In another general aspect, the invention relates to a method of treatinga chronic wound in a subject, wherein the chronic wound has elevatedprotease activity, the method comprising:

-   -   (1) identifying a chronic wound as having elevated protease        activity; and    -   (2) applying a human amniotic fluid or a processed human        amniotic fluid comprising a therapeutically effective amount of        tissue inhibitors of matrix metalloproteinases to the chronic        wound identified as having elevated protease activity, thereby        treating the chronic wound.

In a preferred embodiment, the step of identifying the chronic wound ashaving elevated protease activity comprises detecting concentration oractivity of one or more matrix metalloproteinases (MMPs) in the wound.

In yet another general aspect, the invention relates to a wound dressingcomprising a human amniotic fluid or a processed human amniotic fluidcomprising a therapeutically effective amount of tissue inhibitors ofmatrix metalloproteinases. Embodiments of the invention also relate tomethods of treating chronic wounds comprising applying a wound dressingof the invention to the chronic wound, and preferably to a chronic woundhaving elevated protease activity.

Other aspects, features and advantages of the invention will be apparentfrom the following disclosure, including the detailed description of theinvention and its preferred embodiments and the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles and patents are cited or described in thebackground and throughout the specification; each of these references isherein incorporated by reference in its entirety. Discussion ofdocuments, acts, materials, devices, articles or the like which has beenincluded in the present specification is for the purpose of providingcontext for the invention. Such discussion is not an admission that anyor all of these matters form part of the prior art with respect to anyinventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention pertains. In this application, certainterms are used, which shall have the meanings as set in thespecification. It must be noted that as used herein and in the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise.

It is now discovered that human amniotic fluid and processed humanamniotic fluid comprising a therapeutically effective amount of tissueinhibitors of matrix metalloproteinases is effective in treating chronicwounds, and particularly chronic wounds characterized as having elevatedprotease activity.

As used herein, the term “subject” means any animal, and preferably amammal. Examples of mammals include, but are not limited to, cows,horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs,monkeys, humans etc., more preferably a human.

As used herein, the term “donor” refers to a female subject from whomamniotic fluid will be or has been obtained. The donor can be anyanimal, preferably a mammal, and is most preferably a human.

As used herein, the term “chronic wound” refers to a wound that does notproceed through the normal repair process of wound healing. The normalrepair process of wound healing typically includes four stages: (1)clotting/hemostasis stage; (2) inflammatory stage; (3) tissue cellproliferation stage; and (4) tissue cell remodeling stage. Chronicwounds that do not proceed through the normal repair process of woundhealing can stall in one or more stages, inefficiently or ineffectivelycomplete one or more stages, skip one or more stages, or never reach oneor more stages of the wound healing process. The term “chronic wound” isalso intended to encompass wounds that fail to respond to conventionaltreatments, and wounds that progress to a non-healing chronic condition.Conditions that slow or stop the wound healing process, thus causingwounds to progress to a non-healing chronic condition include, but arenot limited to, poor blood supply or low oxygen supply to tissues in andaround a wound; infection, e.g., bacterial infections in the wound; aweakened immune system; and tissue swelling. Weakened immune system canbe caused by diseases, such as diabetes and cancer, or poor health andnutrition.

The term “chronic wound” also refers to a wound that does not healcompletely within three months. However, wounds that do not heal withinthirty days and are being treated according to normal treatmentprocedures are also considered chronic wounds (see, e.g., the 2005assessment of chronic wound treatment technologies form the Centers forMedicare and Medicaid Services (CMS)).

Non-limiting examples of chronic wounds include ulcers (e.g., diabetic,venous, decubitus (pressure)); non-healing surgical wounds, traumawounds, burns, and amputation wounds; and infected tissue compromised bya weakened immune system.

As used herein, the term “ulcer” refers to a break in the skin or amucous membrane evident by a loss of surface tissue, tissuedisintegration, necrosis of epithelial tissue, nerve damage, and/or pus.Typical examples of ulcers include skin ulcers, such as venous ulcers,diabetic ulcers (e.g., diabetic foot ulcer), and decubitus (pressure)ulcers. Venous ulcers usually occur in the legs, and are often theresult of improper blood flow and/or restriction in blood flow, leadingto tissue damage that causes the wound. Decubitus or pressure ulcers,more commonly referred to as bed sores, are caused by ischemia thatoccurs when pressure on the tissue is greater than the blood pressure inthe capillaries at the wound site, thus restricting blood flow into thearea. Pressure ulcers most commonly occur in individuals with limitedmobility or paralysis, which inhibits the movement of body parts thatare subjected to pressure. Diabetic ulcers, such as diabetic footulcers, are open sores or wounds that usually develop on the bottom ofthe foot of diabetes patients.

Trauma wounds can progress to chronic wound status due to infection orinvolvement of other factors within the wound bed that inhibit properhealing. Chronic wounds can also arise from burn treatment and relatedskin grafting procedures that do not proceed through the normal repairprocess of wound healing. In various types of burns, ulcers, andamputation wounds, skin grafting may be required. In certain instances,patients with ischemia or poor vascularity can experience difficulty inthe graft “taking,” which can stall the healing process and lead to theformation of chronic wounds in and around the grafting area.

Chronic wound formation can also occur in patients where the risk ofinfection is high due to a weakened immune system, such as tissueimpacted by radiation, patients undergoing cancer treatments, patientsaffected by immune compromised diseases such as HIV/AIDS, etc. In suchpatients with a weakened immune system, inflammation of a wound can beprolonged, thereby interfering with the wound healing process andincreasing the susceptibility of wounds to develop into chronic wounds.

As used herein, the terms “treat,” “treating,” and “treatment” are allintended to refer to an amelioration or reversal of at least onemeasurable physical parameter related to a chronic wound, which is notnecessarily discernible in the subject. As an illustrative andnon-limiting example, the surface area of the chronic wound is ameasurable physical parameter that can be determined before and aftertreatment to evaluate the effect of the treatment on the chronic wound.The terms “treat,” “treating,” and “treatment” can refer to improvingthe progression of wound healing, such as increasing the rate of woundhealing. In one embodiment, “treat,” “treating,” and “treatment” referto a reduction or an alleviation of one more symptoms associated withchronic wounds, such as pus in and around the wound area; bleeding,swelling, or pain in the wound area; trouble moving the area affected bythe wound; deepening and/or enlargement of the wound; and dark or blackskin around the wound, particularly dark or black skin that is warm tothe touch. In another embodiment, “treat,” “treating,” and “treatment”refer to partial or complete healing of a chronic wound. In yet anotherembodiment, “treat,” “treating,” and “treatment” refer to promotingproliferation of the epithelial cells that grow over the wound bed tocover it. And in yet another embodiment, “treat,” “treating,” and“treatment” refer to restoring the progression of a disturbed woundhealing process to the normal stages of the repair process.

“Matrix metalloproteinases” and “MMPs” refer to a family ofzinc-dependent endopeptidases. MMPs are produced by inflammatory cells(e.g., neutrophils and macrophages) and wound cells (e.g., epithelialcells, fibroblasts, vascular endothelial cells), and play a role in manybiological processes, including cell proliferation, inflammation, andwound healing. MMPs are first produced and released in an inactive formknown as the “pro-MMP” form. Inactive pro-MMPs are subsequentlyactivated by proteases that cleave off a portion of the protein.Examples of MMPs include, but are not limited to, MMP-1, MMP-2, MMP-3,MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, and MMP-9. Although the specificnatural substrates for each of the MMPs varies, MMPs are generallycapable of degrading extracellular matrix proteins, such as collagen,gelatin, proteoglycans, etc.

As used herein, “tissue inhibitor of matrix metalloproteinases” and“TIMPs” refer to a family of protease inhibitors that modulate theactivity of matrix metalloproteinases. The human genome encodes fourTIMPs: TIMP1, TIMP2, TIMP3, and TIMP4. TIMPs modulate matrixmetalloproteinases by inhibiting the activity of activated MMPs, or byblocking the activation of pro-MMPs. According to embodiments of theinvention, an amniotic fluid comprising a therapeutically effectiveamount of TIMPs can affect the activity of MMPs in a wound by inhibitingthe activity of activated MMPs, or by blocking the activation ofpro-MMPs.

As used herein, “elevated protease activity” and “EPA,” when used withreference to a chronic wound, are intended to refer to an increasedlevel of enzymatic activity of certain proteases present within thewound. Examples of proteases that can have elevated activity within awound include matrix metalloproteinases and serine proteases, such asplasmin, neutrophil elastase, cell chymase, etc. A wound characterizedas having an elevated protease activity, or that is referred to as an“EPA wound,” is a wound in which the enzymatic activity level ofproteases, particularly matrix metalloproteinases, is increased ascompared to the activity detected of the same proteases in a wound inwhich healing is progressing according to the normal repair process. Theincreased enzymatic activity detected can be the result of an increasedconcentration or amount of proteases in the wound, or it can be due toincreased specific activity of the proteases. For example, increasedenzymatic activity of MMPs detected in a wound can result from anincreased amount of MMPs present in the wound; an increased amount ofMIMPs present in the activated form, rather than the pro-MMP inactiveform; and/or an increased amount of MMPs uninhibited by TIMPs, any ofwhich can lead to a higher detectable level of enzymatic activity in thewound.

The term “therapeutically effective amount” is intended to refer to anamount of an active ingredient or component that elicits the desiredbiological or medicinal response in a subject. As used herein withreference to tissue inhibitors of matrix metalloproteinases (TIMPs), atherapeutically effective amount means an amount of TIMPs that resultsin treatment of a chronic wound in a subject, including improving theprogression of wound healing, reducing or alleviating one or moresymptoms associated with chronic wounds, partial or complete healing ofthe chronic wound, promoting proliferation of epithelial cells that growover and cover the wound bed, and restoring the progression of adisturbed wound healing process to the normal states of the repairprocess. One of ordinary skill in the art will recognize that thetherapeutically effective amount of TIMPs to be used in the inventioncan vary with factors, such as the particular subject, (e.g., age, diet,health, etc.), type of chronic wound, severity of the chronic wound, andany underlying complications or conditions in the subject that slow orstop the wound healing process. For example, many chronic wound patientshave one or more underlying complications or conditions that can affectthe wound healing process and thus subsequent treatment of the wound,such as diabetes, smoking, vascular disease, obesity, auto-immunedisease and the like.

The invention provides methods of treating chronic wounds in a subjectcomprising applying a human amniotic fluid or a processed human amnioticfluid to the chronic wound. According to embodiments of the invention,the amniotic fluid comprises a therapeutically effective amount oftissue inhibitors of matrix metalloproteinases (TIMPs), which caninhibit proteases in the wound that interfere with the wound repairprocess, thereby facilitating wound healing. Amniotic fluid naturallycontains TIMPs, as well as many other regenerative components that areefficacious for wound healing, such as growth factors, stem cells,protease inhibitors, etc. Accordingly, providing a therapeuticallyeffective amount of TIMPs in an amniotic fluid can further enhancehealing of chronic wounds as a result of the other components present inthe amniotic fluid.

During gestation of an embryo, the body creates a placental sac made ofamnion and chorion tissues surrounding the fetus and containing amnioticfluid to both protect and nourish the fetus. A purpose of TIMPs inamniotic fluid is to prevent the amnion and chorion tissues from beingprematurely degraded by proteases, as such degradation would causepremature rupture of the placental sac and pre-term birth of the fetus.However, once the pregnancy has reached full term, TIMP levels in theamniotic fluid typically will fall abruptly, which allows fordegradation of the amnion and chorion tissues, release of the amnioticfluid, and subsequent birth of the fetus. Thus, TIMP levels in amnioticfluid are typically at their highest during the late pre-term period ofpregnancy, which prevents degradation of the amnion and chorion tissues,thus maintaining the integrity of the placental sac.

According to embodiments of the invention, the therapeutically effectiveamount of TIMPs can be provided by the natural level of TIMPsendogenously present in the amniotic fluid, or the therapeuticallyeffective amount of TIMPs can be provided by supplementing the amnioticfluid with endogenous TIMPs. Supplementing the amniotic fluid collectedfrom the female donor with exogenous TIMPs, e.g., purified TIMPs, canincrease the effective concentration of TIMPs in the amniotic fluid andfurther enhance its effects on wound healing.

In a preferred embodiment, the amniotic fluid is a processed amnioticfluid, more preferably a processed human amniotic fluid. As used herein,the term “processed amniotic fluid” is an amniotic fluid that has beenmanipulated in some way after being collected from the donor. Forexample, the amniotic fluid collected from the donor can be concentratedto remove water from the amniotic fluid, thus concentrating manyamniotic fluid components, including TIMPs. Concentrating amniotic fluidcan increase the concentration of TIMPs and/or other components in theamniotic fluid, thus achieving optimal therapeutic concentrations ofTIMPs and other components in the amniotic fluid, which can furtherenhance wound healing. However, the invention is not limited toprocessing amniotic fluid by removing water to concentrate TIMPs, andother methods can be used to optimize the concentration of TIMPs in theamniotic fluid. For example, the amniotic fluid can also be processed byexogenously adding one or more components, e.g., TIMPs to the amnioticfluid, thus increasing the concentration of TIMPs.

According to embodiments of the invention, the amniotic fluid can beobtained from a human female donor during gestation at a point when TIMPlevels are typically near or at their maximum. As known by one ofordinary skill in the art, the gestation period in human females isdivided into three trimesters: the first trimester (weeks one throughtwelve), the second trimester (weeks thirteen through twenty-seven), andthe third trimester (weeks twenty-eight to birth). Gestation in humanfemales is also characterized according to terms, i.e., specific periodsof gestation during which birth occurs: pre-term (before 37 weeks ofgestation), early term (37 weeks to 38 weeks, 6 days of gestation), fullterm (39 weeks to 40 weeks, 6 days of gestation), and late term (41weeks to 41 weeks 6 days of gestation). TIMP levels have beendemonstrated to fall rapidly in amniotic fluid at birth. Therefore, in apreferred embodiment, the amniotic fluid is obtained at a point justprior to birth.

According to embodiments of the invention, human amniotic fluid for usein the invention can be obtained from a human female donor. Preferably,the amniotic fluid is obtained at the time of Caesarean sectiondelivery. When the Caesarean section delivery is elective, collection ofthe amniotic fluid can occur during the late pre-term period ofpregnancy, i.e., during weeks 28 to 37 of gestation, and preferablycloser to week 37 of gestation, when TIMP levels are typically at ornear their maximum. For example, the amniotic fluid can be obtainedduring week 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 of gestation.

According to embodiments of the invention, the method of treating achronic wound can further comprise determining the concentration ofTIMPs in the amniotic fluid, thereby verifying that the amniotic fluidhas an increased concentration of TIMPs. Any method known in the art inview of the present disclosure can be used to determine theconcentration of TIMPs, such as enzyme-linked immunoabsorbent assay(ELISA), Bradford assay, electrophoresis techniques (e.g., SDS-PAGE,Western blot), etc. For example, the concentration of TIMPs can bedetermined by testing a sample of amniotic fluid obtained byamniocentesis or at the time of the Caesarean section delivery.

The amniotic fluid can be further processed so that it has a relativelyhigh viscosity for ease of application and for remaining in the desiredarea after the application. For example, the amniotic fluid can beconcentrated to remove water by any technique known to those skilled inthe art in view of the present disclosure including, but not limited to,dialysis, diffusion techniques, centrifugation, lyophilization, andvacuum filtration. The amniotic fluid can be further treated in order topromote preservation, lengthen shelf life, or improve stability. Thesetreatments include, but are not limited to, sterilization, e.g.,gamma-irradiation; cooling, refrigeration, and freezing; and addition ofone or more excipients. Examples of excipients that can be included inthe amniotic fluid include, but are not limited to, preservatives;cryopreservatives; antimicrobial agents and other substances to preventthe growth of microbes, such as antimicrobial and antiviral agents;thickeners; salts; colorants; and agents that improve the viscosity ofthe composition.

According to embodiments of the present invention, the amniotic fluidcan further comprise a cryoprotectant. Any cryoprotectant suitable forpharmaceutical use known to those skilled in the art in view of thepresent disclosure can be used including, but not limited to dimethylsulfoxide (DMSO), sucrose, glycerol, glucose, and any other sugars,e.g., monosaccharides or disaccharides, alcohols and penetrating agents,or combinations thereof.

Preferably, the amniotic fluid is free of particulate matter, such ascellular debris and tissue debris. Particulate matter can be removedfrom the amniotic fluid by any method known in the art for removingparticulate matter from biological samples, including but not limited tofiltration and centrifugation. Particulate matter can be removed at anytime after the amniotic fluid has been collected from the donor.Preferably, the particulate material is removed prior to any otherprocessing or treatment steps.

For example, using an ultrafiltration approach, a semi-permeablecontainer is filled with raw amniotic fluid, and then a pressuregradient is applied across the semi-permeable membrane using any numberof techniques known to those skilled in the art including, but notlimited to, a high permeability dialyzer. As another illustrativeexample, when employing hemodialysis techniques, an electrolyte solution(dialysate) can be applied on one side of a membrane, creating aconcentration gradient, which causes water and other non-proteincellular components of the amniotic fluid to flow through thesemi-permeable membrane. As yet another illustrative example, rapidultrafiltration approaches can be used. Rapid ultrafiltration approachesemploy a semi-permeable membrane cylindrical container that rotatesconstantly in order to avoid filter clogging even as a pressure gradientis applied to the contained fluid—either from within the container(pushing), or from the opposite side of the semi-permeable membrane(pulling). The amniotic fluid can also be concentrated by removal ofwater using any technique known to those of ordinary skill in the art.For example, substantially all of the water can be removed bylyophilization, or the amount of water can simply be reduced by vacuumfiltration. Other methods that can be used to process the amniotic fluidare described in U.S. patent application Ser. No. 14/950,186, which isherein incorporated by reference in its entirety.

According to embodiments of the invention, any chronic wound in view ofthe present disclosure can be treated by the methods described herein.In a preferred embodiment, the chronic wound is identified as havingelevated protease activity. Preferred examples of chronic wounds to betreated by the methods of the invention include ulcers, preferably skinulcers, such as diabetic ulcers (e.g., diabetic foot ulcer), venousulcers, and pressure ulcers, and particularly preferred examples ofchronic wounds are elevated protease activity skin ulcers, such asdiabetic ulcers (e.g., diabetic foot ulcer), venous ulcers, and pressureulcers. Other examples of chronic wounds to be treated by methods of theinvention include infected wounds in patients with a compromised immunesystem; non-healing surgical wounds, trauma wounds, and burn wounds;wounds that have progressed to a non-healing chronic state as a resultof poor blood supply or low oxygen supply to tissues in and around awound, infection, or tissue swelling; and wounds from skin graftfailure.

In certain embodiments of the invention, a chronic wound to be treatedis a chronic wound having elevated protease activity (EPA), particularlyelevated activity of matrix metalloproteinases (MMPs). Particular MMPsthat are known to play a role in wound healing and/or inflammationinclude MMP-1, MMP-2, MMP-8, and MMP-9. When MMPs are present in a woundat too high a level, they begin to degrade proteins that are not theirnormal substrates, which can result in degradation of proteins that areessential for wound healing, such as extracellular matrix proteins, thusimpairing the healing process.

In a preferred embodiment of the invention, the chronic wound to betreated has elevated activity of one or more MMPs, particularly one ormore MMPs selected from the group consisting of MMP-1, MMP-2, MMP-8, andMMP-9.

A method of treating a chronic wound according to an embodiment of theinvention can further comprise a step of identifying a chronic wound ashaving elevated protease activity. Any method known in the art in viewof the present disclosure can be used to determine if a chronic woundhas elevated protease activity. For example, biological samples, such asfluid or swabs, can be taken directly from the wound bed to be testedfor elevated protease activity. The biological samples can then beanalyzed using in vitro techniques to qualitatively or quantitativelydetect or measure the enzymatic activity level or concentration of theproteases of interest. Any method or assay known in the art in view ofthe present disclosure can be used to detect or measure enzymaticactivity or concentration, including, but not limited to,immunochromatographic methods, colorimetric assays, fluorescence-basedassays, ELISA, Bradford assay, electrophoresis techniques (e.g.,SDS-PAGE, Western blot), etc.

In particular embodiments of the invention, in vitro assays designed tospecifically detect MMP activity can be used to analyze the biologicalsamples taken from the wound. The in vitro assays can be used at pointof care. Commercially available kits and tests for measuring elevatedprotease activity in wounds, as well as commercially available kits andtests for specifically measuring MMP activity can also be used with amethod of the invention. Such kits and tests can be used according tothe manufacturer's instructions.

According to embodiments of the invention, the amniotic fluid can beadministered to a chronic wound alone or in combination with one or moreactive agents. Examples of active agents that can be administered withthe amniotic fluid include, but are not limited to, morphogenicproteins, small molecule compounds, pharmaceutical agents,anti-microbial agents, anti-inflammatory agent, agents that preventscarring and/or adhesions, and analgesics. Administering the amnioticfluid in combination with such an active agent may further enhance thewound healing process by promoting epithelial cell proliferation,decreasing inflammation, reducing or preventing infection, alleviatingpain, etc.

The chronic wound can be cleansed prior to application of the amnioticfluid. For example, the wound can be washed with water or a sterilesaline solution. The wound can also be treated by surgical or mechanicaldebridement to remove dead tissue, dirt, or other objects that can delayhealing and lead to infection.

According to embodiments of the invention, the amniotic fluid can beapplied as a single application to a wound, or it can be appliedmultiple times, e.g., in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 applications,etc. The amniotic fluid can be applied once a day, or multiple times aday. The amniotic fluid can be continually re-applied to the wound for aperiod of a few days, a few weeks, or a few months, and is preferablyreapplied until complete healing of the wound is observed. The optimalnumber, frequency, and duration of applications of the amniotic fluidwill depend upon various factors including the particular type of wound,how long the chronic wound has been persisting, the underlying cause ofthe chronic wound, etc. One of ordinary skill in the art will be able todetermine the frequency and duration of applications of the amnioticfluid in view of the above factors in order to achieve the desiredoutcome.

The invention also relates to a wound dressing comprising a humanamniotic fluid or a processed human amniotic fluid comprising atherapeutically effective amount of tissue inhibitors of matrixmetalloproteinases. The term “wound dressing” as used herein refers to amaterial used for covering or protecting a wound. For example, a wounddressing can be a surgical dressing, a bandage, or any other materialsuitable for covering or protecting a wound.

According to embodiments of the invention, the wound dressing can beabsorbed with the human amniotic fluid. The wound dressing can compriseany amniotic fluid in view of the present disclosure.

The wound dressing can be made of any material suitable for covering achronic wound. Examples of suitable materials include, but are notlimited to, any medical grade polymer or monomer, polyvinyl alcohol(PVA), polytetrafluoroethylene (PTFE), expanded polytetrafluoroethylene(ePTFE), porous polyethylene, porous polypropylenes, etc. Applying anamniotic fluid comprising a therapeutically effective amount of TIMPsabsorbed in a wound dressing can further enhance the wound healingprocess by providing a protective covering to the chronic wound, whichcan prevent infection, keep the wound clean, etc.

According to embodiments of the invention, the wound dressing or bandagecan be used in a method of treating a chronic wound of the invention.The method comprises applying the wound dressing to a chronic wound,thereby treating the chronic wound. Preferably, the chronic wound is achronic wound having elevated protease activity. Thus, in a preferredembodiment, the method further comprises a step of identifying a chronicwound as a wound having elevated protease activity prior to applying thewound dressing to the chronic wound. A wound dressing according toembodiments of the invention can be used in any of the methods describedherein.

Embodiments of the invention also relate to harvesting TIMPs fromamniotic fluid obtained from the donor, preferably amniotic fluidobtained from the donor at a point during gestation when the level ofTIMPs is at or near the maximum. TIMPs harvested from the amniotic fluidcan be purified and formulated into a pharmaceutical compositionsuitable for application to a chronic wound. TIMPs harvested from theamniotic fluid can also be used to supplement an amniotic fluid prior toapplication of the amniotic fluid to a chronic wound. TIMPs can beharvested from amniotic fluid by any method known in the art forpurifying and isolating proteins from biological mixtures in view of thepresent disclosure, such as size-exclusion chromatography,ultracentrifugation, affinity chromatography, or other chromatographicseparation methods, etc.

In one embodiment of the invention, a method of treating a chronic woundin subject comprises applying a pharmaceutical composition comprising atherapeutically effective amount of TIMPs to the chronic wound, therebytreating the chronic wound. The method can further comprise identifyinga chronic wound as having elevated protease activity, such as elevatedprotease activity of one or more MMPs, e.g., MMP-1, MMP-2, MMP-8, MMP-9,etc. Any chronic wound in view of the present disclosure can be treatedby applying a pharmaceutical composition comprising a therapeuticallyeffective amount of TIMPs according to a method of the invention.

Methods of obtaining an amniotic fluid from a donor using theappropriate sterile techniques are well known to those of ordinary skillin the art. One of ordinary skill in the art is also familiar withprocedures for safely and humanely obtaining an amniotic fluid from adonor in an aseptic manner. For example, human amniotic fluid can beobtained from a donor who is undergoing an amniocentesis procedure,Caesarean section delivery, or vaginal birth using a specially designedreceptacle to collect the fluid. Preferably, the amniotic fluid isobtained from a donor undergoing a Caesarean section delivery, and ismore preferably obtained from a donor undergoing a pre-term Caesareansection delivery, i.e., prior to week 37 of gestation. Amniotic fluidobtained from a donor undergoing vaginal birth, or from an amniocentesisprocedure can also be used with a method of the invention, however alarger quantity of amniotic fluid can be obtained from a donorundergoing a Caesarean section delivery, and is thus the preferredmethod for obtaining amniotic fluid. Also, amniotic fluid at the time ofvaginal delivery at term typically has lower levels of TIMPs.

According to embodiments of the invention, amniotic fluid used in theinvention is procured from a female donor. Informed consent is obtainedfrom the female donor by following guidelines as promulgated by theAmerican Association of Tissue Banks and consistent with guidelinesprovided by the Food and Drug Administration: a federal agency in theDepartment of Health and Human Services established to regulate therelease of new medical products and, finally, if required by anestablished review body of the participating hospitals or institutions.The female donor is informed that she will be subject to risk assessmentto determine if she is qualified as an amniotic fluid donor. She willalso be informed of the tests for the risk assessment. The female donoris further informed that, if she is selected as an amniotic fluid donorbased on the risk assessment, her birth tissues, such as placenta andamniotic fluid, may be collected at birth, tested and processed formedical uses. The informed consent includes consent for risk assessmentand consent for donation of birth tissues and amniotic fluid.

Risk assessment is conducted on the female donor with informed consentto evaluate her risk factors for communicable diseases, such as humanimmunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus(HCV), cytomegalovirus (CMV), human T-lymphotropic virus (HTLV),syphilis, etc., as is required by regulating bodies. Medical and socialhistories of the female donor, including physical exam record, and/orrisk assessment questionnaire, are reviewed. Those with high riskfactors for the communicable diseases are excluded.

Consent to draw blood at the time of delivery and 1 to 12 monthspost-delivery is obtained from female donors with low risk factors forthe communicable diseases. Screening tests on communicable diseases,such as HIV 1 and 2, HCV, HbCore, syphilis, HTLV I/II, CMV, hepatitis Band C, are conducted by conventional serological tests on the bloodsample obtained at birth. The initial screening tests are preferablycompleted within 7 days after birth. Preferably, the screening tests areconducted again on a second blood sample collected a few monthspost-delivery, to verify the previous screening results and to allow fordetection of communicable disease acquired shortly before birth, but areshown as “negative” on the previous screening tests. The second bloodsample can be collected 1-12 months, preferably 6 months, post birth.

Only female donors with informed consent who are tested negative for thecommunicable diseases are approved as amniotic fluid donors. In apreferred embodiment, only female donors with informed consent who aretested negative for the communicable diseases in both screening testswith the blood sample drawn at birth and the blood sample drawn 6 monthspost-delivery are approved as amniotic fluid donors.

In order for a wound to heal, extracellular matrix need not only be laiddown over the wound, but the extracellular matrix must also undergodegradation and remodeling to form a mature tissue. Proteases,particularly matrix metalloproteinases (MMPs), are known to degradeextracellular matrix components, and during normal wound healing, MMPactivity is appropriately balanced by endogenous protease inhibitors. Incontrast, in chronic wounds, the balance between MMP and the activity ofendogenous protease inhibitors is disturbed, which can result in anincreased activity or concentration of MMPs. Accordingly, and withoutwishing to be bound by any theories, it is believed that the highconcentration of TIMPs in amniotic fluid near term can inhibit theactivity of proteases having elevated activity in chronic wounds,particularly MMPs, thereby facilitating the healing of chronic wounds.

The following examples of the present invention are to furtherillustrate the nature of the invention. It should be understood that thefollowing examples do not limit the invention and that the scope of theinvention is to be determined by the appended claims.

EXAMPLES Example 1 Treatment of a Chronic Wound with Human AmnioticFluid Comprising TIMPs

Amniotic fluid is isolated from a pregnant human female donor undergoinga pre-term Caesarean section delivery at week 36 of gestation. Theisolated amniotic fluid is processed by concentrating the fluid toremove excess water, thus increasing the effective concentration ofendogenous TIMPs in the amniotic fluid.

The processed amniotic fluid is applied directly to a human patienthaving a chronic leg ulcer. The processed human amniotic fluid isapplied at least once per day. Healing of the ulcer is monitored atleast by observing the boundaries of the wound. The processed humanamniotic fluid is continually applied to the ulcer at least once a dayuntil the ulcer is healed.

Example 2 Treatment of a Chronic wound with Human Amniotic FluidComprising TIMPs

Amniotic fluid is isolated from a pregnant human female donor undergoinga pre-term Caesarean section delivery at week 36 of gestation. Theisolated amniotic fluid is processed by concentrating the fluid toremove excess water, thus increasing the effective concentration ofendogenous TIMPs in the amniotic fluid. Purified TIMPs are added to theprocessed amniotic fluid to further increase the concentration of TIMPs.

The processed amniotic fluid is applied directly to a human patienthaving a chronic leg ulcer. The processed human amniotic fluid isapplied at least once per day. Healing of the ulcer is monitored byobserving the boundaries of the wound. The processed human amnioticfluid is continually applied at least once a day until the ulcer ishealed.

Example 3 Treatment of a Chronic Wound with Human Amniotic FluidComprising TIMPs

Amniotic fluid is isolated from a pregnant human female donor undergoinga pre-term Caesarean section delivery at week 36 of gestation. Theisolated amniotic fluid is processed by concentrating the fluid toremove excess water, thus increasing the effective concentration ofendogenous TIMPs in the amniotic fluid.

A leg ulcer in a patient is identified as a chronic wound havingelevated protease activity by using a commercially available kit thatmeasures MMP activity according to the manufacturer's instructions. Theprocessed amniotic fluid is applied to the leg ulcer at least once a dayuntil the ulcer is healed.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

REFERENCES

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We claim:
 1. A method of treating a chronic wound in a subjectcomprising applying a human amniotic fluid or a processed human amnioticfluid comprising a therapeutically effective amount of tissue inhibitorsof matrix metalloproteinases (TIMPs) to the chronic wound, therebytreating the chronic wound.
 2. The method according to claim 1, whereinthe amniotic fluid is obtained from a female donor between week 28 andweek 37 of gestation.
 3. The method according to claim 1, wherein theamniotic fluid is obtained from a female donor undergoing a Caesareansection procedure, wherein the Caesarean section procedure takes placeat a point during gestation near term.
 4. The method according to claim1, wherein the chronic wound is characterized as having elevatedprotease activity.
 5. The method according to claim 4, wherein thechronic wound comprises elevated protease activity of one or more matrixmetalloproteinases (MMPs).
 6. The method according to claim 5, whereinthe one or more MMPs is selected from the group consisting of MMP-1,MMP-2, MMP-8, and MMP-9.
 7. The method according to claim 1, wherein thechronic wound is selected from the group consisting of an ulcer; anon-healing surgical wound, burn, trauma wound, or amputation wound; afailed skin graft; and an infected tissue or wound compromised by aweakened immune system in the subject.
 8. The method according to claim1, wherein the chronic wound is an ulcer selected from the groupconsisting of diabetic ulcer, diabetic foot ulcer, venous ulcer, venousleg ulcer, and decubitus (pressure) ulcer.
 9. The method according toclaim 1, wherein the amniotic fluid is administered in combination withone or more active agents selected from the group consisting ofmorphogenic proteins, small molecule compounds, pharmaceutical agents,anti-microbial agents, anti-inflammatory agent, agents that preventscarring and/or adhesions, and analgesics.
 10. The method according toclaim 1, wherein the amniotic fluid is supplemented with exogenouslyadded TIMPs.
 11. A method of treating a chronic wound in a subject,wherein the chronic wound has elevated protease activity, the methodcomprising: (1) identifying a chronic wound as having elevated proteaseactivity; and (2) applying a human amniotic fluid or a processed humanamniotic fluid comprising a therapeutically effective amount of tissueinhibitors of matrix metalloproteinases to the chronic wound identifiedas having elevated protease activity, thereby treating the chronicwound.
 12. The method according to claim 11, wherein the amniotic fluidis obtained from a female donor between week 28 and week 37 ofpregnancy.
 13. The method according to claim 11, wherein the step ofidentifying the chronic wound as having elevated protease activitycomprises detecting activity or concentration of one or more matrixmetalloproteinases (MMPs) in the wound.
 14. The method according toclaim 13, wherein the one or more MMPs is selected from the groupconsisting of MMP-1, MMP-2, MMP-8, and MMP-9.
 15. The method accordingto claim 11, wherein the chronic wound is selected from the groupconsisting of an ulcer; a non-healing surgical wound, burn, traumawound, or amputation wound; a failed skin graft; and an infected tissueor wound compromised by a weakened immune system in the subject.
 16. Themethod according to claim 11, wherein the chronic wound is an ulcerselected from the group consisting of diabetic ulcer, diabetic footulcer, venous ulcer, venous leg ulcer, and decubitus (pressure) ulcer.17. The method according to claim 11, wherein the amniotic fluid isadministered in combination with one or more active agents selected fromthe group consisting of morphogenic proteins, small molecule compounds,pharmaceutical agents, anti-microbial agents, anti-inflammatory agent,agents that prevent scarring and/or adhesions, and analgesics.
 18. Themethod according to claim 11, wherein the amniotic fluid is supplementedwith exogenously added TIMPs.
 19. A wound dressing comprising a humanamniotic fluid or a processed human amniotic fluid comprising atherapeutically effective amount of tissue inhibitors of matrixmetalloproteinases (TIMPs).
 20. A method of treating a chronic wound ina subject comprising applying the wound dressing according to claim 19to the chronic wound, wherein the method optionally comprises a step ofidentifying a chronic wound as having elevated protease activity priorto applying the wound dressing.